Deamino1-phenylalanyl2-arginine8-vasopressin



United States Patent 3,418,307 DEAMIN0 -PHENYLALANYL -ARGININE VASOPRESSIN Roger Boissonnas, Bottmingen, and Ren Huguenin,

Reinach, Basel-Land, Switzerland, assignors to Patented Dec. 24, 1968 when an acid addition salt is desired, the resulting polypeptide I is salified.

The oxidation of the polypeptide V to give the desired final product I is preferably effected with air, oxygen,

Sandal Ltd. (also known as sandal Ag), Base], hydrogen peroxide, potassium ferricyanide or 1, 2-diiodo- Switzerland ethane. An aqueous alcoholic solution or a mixture of No Drawing. Filed Mar. 17, 1966, Ser. No. 535,006 acetone and water may, for example, be used as the Claims priority, application Switzerland, Apr. 15, 1965, aqueous organic ol tion,

1 Cl 0 ,3 4 11 5 The polypeptide of Formula V may be obtained by alm'( splitting off the protective radicals R and R" in one or more stages from an octapeptide derivative of general ABSTRACT OF THE DISCLOSURE Formula IV below, in which R denotes a radical capable The invention relates to new polypeptides from the group consisting of a polypeptide of the formula ITTHC-NH2 CONH: (3H2 NH $5H5 (1351 (3H2 C CECE-$12M CH7 CH2 CH2 (EH2 (3H2 CH1 CH2 CHa-CONH-(5HCONH-CHCONHCHCONH-CIICONHCH-CONCIIC ONHC1EI-C ONHC 112-0 ONII:

CH2 ([3112 t S 1 2 3 4 5 6 7 s 9 Mpr Phe Phe Glu Asp Cys Pro Arg Gly and its acid additional salts. The compounds are pharmaceutically active as exhibited by especially high antidiuretic activity and by unusual and unexpectedly high ratio of antidiuretic to pressoric activity. Pharmaceutical preparations and treatment of diabetes insipidus are also illustrated.

The present invention relates to a new polypeptide and a process for its production.

The present invention provides the polypeptide of Formula I,

and its acid addition salts.

methods in use or described in the literature on the sub- The present invention further provides a process for ject.

the production of compound I and its acid addition salts,

The octapeptide derivative IV may, for example, be

I?TH(3NHz OH:

SIH CHz-CH: $11: CH: CH: CH:

characterized in that a polypeptide of Formula V,

CONH:

CH: CH: CH: CH: CH:

Mpr Phe Phe Glu Asp is oxidized in aqueous or aqueous organic solution at a pH value of 4 to 9, in manner known per se and,

6 7 8 9 Cys P to Arg Gly v produced in that a hexapeptide derivative of general Formula II,

IiIHC-NHR (IIONHa 3H; NH on, CONE: S-R CHz-]CH2 CH; (EH2 CH: (11H: (11H: (1H2 NHrCHC ONHCI-IC ONHCHC ONCHC ONH-CHCONHCH2CONH2 II Glu Asp Oys Pro Arg Gly in which R and R" have the above significance, is con- The selective antidiuretic efl'ect of Compound I could densed with a reactive derivative of a free acid of general not be foreseen from the hitherto known relationships be- Forrnula III, tween chemical structure and biological activity of com- C6115 CBHE pounds with a similar structure, as the removal of the free 1 CH CH amino radical in the 1-position (Deamino -Arg -vaso- 2 l 2 I 2 pressin) and of the phenolic hydroxy radical in the 2-posi- (3-011 tion (Phe -Arg -vasopressin) did not substantially increase Mpr Phe Phe the selectivity.

Mpr=fl-1tlercapto-propiouyl- III The dosage of Compound I should be adapted to the individual degree of hormone deficiency which may vary considerably and has a range of 0.1 to 5 IU administered 3 to 4 times daily subcutaneously or intramuscularly.

The polypeptide of the invention may be used in the form of a free base or a salt of an organic or inorganic acid or an acid radical containing polymer (e.g., alginic acid, carboxymethyl cellulose, tannic acid), either as a pharmaceutical on its own or in the form of appropriate medicinal preparations, e.g. for oral, parenteral, enteral or intranasal administration. In order to produce suitable medicinal preparations the compounds are worked up with inorganic or organic adjuvants which are inert and physiologically acceptable. Examples of such adjuvants are:

in which R has the above significance.

Examples of radicals for protecting the sulfhydryl radical in the above process by temporarily blocking it are the benzyl or p-xylyl radicals, while examples of radicals for protecting the guanido radical are the p-toluenesulfonyl or benzenesulfonyl radicals.

It should be noted that the octapeptide derivative IV need not be produced in the manner described above as it may also be produced by condensation of two polypeptides other than the ones indicated above (or a polypeptide and an amino acid) in the form of their protected derivatives, or an octapeptide and S-mercaptopropionic acid in the form of their protected derivatives.

i Starting Platefials for the Production of the oeta- For tablets: lactose, starch, talc and stearic acid; P P deflvatlve Insofar as y are unknown, are For injectable solutions: water, alcohols, glycerin and produced by known methods for the synthesis of peptides, vegetable oil it being possible to join together the amino acids and ,B-mercaptopropionic acid one at a time or by first forming For suppositories: natural or hardened oils and waxes; For intranasal sprays: water, glycerin and other liquid eencstiment I PI fi i g and l ll lf g g i- Ph 2 substances tolerated by the mucous membrane.

ompoun w c may e ca e eammo e Arg -vasopressin, has antidiuretic eifect which is consider- P P f y furtkfel'mel'e eontamfillltable P ably stronger than that of natural human vasopressin, serving, stabrhzrng and wetting agents, solubihzers, sweet- Arg -vasopressin of Formula VI, enmg and colorlng substances and flavorrngs.

IfH-C-NH: GoNHl OH: NH

(fgHsOH (ljsH5 (IJHZ CONE: CHz-CH: CH: $11: (3H2 $117 CH9 $1512 (3H2 NHroH-oo-NH-oH-o ONH-CHCONHCHCO-NHCH-C o-NHJfH-G ONOH-OO-NH-CH-CO-NH-CHz-O ONH:

?H2 $1312 VI S S Arginine Vasopressin 1 2 3 4 5 6 7 s 9 Cys Tyr Phe Glu Asp Oys Pro Arg Gly from which the new compound differs in that it has a In the following non-limitative examples all temperafl-mercaptopropionyl-L-phenylalanyl radical in place of tures are indicatedin degrees centigrade. :gdlgsenn-cystrnyl-L-tyrosyl radical 1n the positions 1 EXAMPLE 1: POLYPEPTIDE I Upon comparing the elTects expressed in International (a) figfgg gg f g i acid 2,4,5413' Units (IU) it may be seen that natural arginine vasoc my 6 e pressin has a pressoric eifect which is as strong as its anti- 20 Y 'l PP P P P aeld and 22 8- diuretic efi'ect, whereas Compound I has a pressonc efiect 0f 2,4,5-tf1eh10I0PheIl01 are d 1S$P1ved Q of ethyl which amounts to only approximately one-thirtieth of its acetate and 20 1111- Of e eoollng 1S e e at antidlffietlc effect. 'Ilfie antidigretifc effect in colmparison 31 fifl l l -h i y g l l y t are wit e pressoric e ect is t ere ore more se ective in a e e 80 11 10!! 18 S 3 e11 Ours a room P Compound 1 th i t l i i vasopi-essin D ature, the dicyclohexylurea precipitate is filtered with sueto thi property Compound I i especially i di t d f tion and the filtrate is evaporated in a vacuum at 30. use in the treatment of Diabetes insipidus. The residue is dissolved in ethyl acetate and the solution is washed with aqueous sodium bicarbonate and then with Antidi- Pressoric R elationwater. After drying over sodium sulfate the ethyl acetate Compound 23 fi i 2 g gs g is evaporated. The residue is left to stand at 10 whereby IU/mg. tivity) S-benzyl-B-mercaptopropionic acid 2,4,5-trichlorophenyl Deammo LPhehArgaflasopressm ester, having a melting point of 17, crystallizes. l$2$33a111111111111111111: i 3%? 1 y -p y nine- Deamino Arg -vasopressin 1, 300:1:200 370:1:20 3.5 meth l t r Phe Arg -vasopressin 350 122i13 2. 9

47 g. of L-phenylalaninemethyl ester in 200 cc. of dimethylformamide are added to 122 g. of N-carbobenzoxy- L-phenylalanine 2,4,5-trichlorophenyl ester. After 16 hours dilution is effected with 1200 cc. of ethyl acetate. The solution is washed with N hydrochloric acid, aqueous sodium bicarbonate and water. After drying over sodium sulfate, evaporating the ethyl acetate and recrystallizing the residue from chloroform/ether, N-carbobenzoxy-L- phenylalanine methyl ester, having a melting point of 149, [a] =-18 (dimethylformamide), is obtained.

(c) S-benzyl-fi-mercaptopropionyl-L-phenylalanyl-L- phenylalanine methyl ester 74 g. of N-carbobenzoxy-L-phenylalanyl-L-phenylalanine methyl ester are dissolved in 400 cc. of anhydrous acetic acid which have been saturated with hydrogen bromide. The solution is left to stand for one hour at 20, evaporation is effected in a vacuum below 40 and the residue is carefully Washed with diethyl ether. The residue is subsequently dissolved in 360 ml. of water at 0, 22 g. of potassium carbonate are added and extraction is effected with ethyl acetate at 0. After drying over sodium sulfate, 55 g. of S-benzyl-B-mercaptopropionic acid, 2,4,5-trichlorophenyl ester are added and the mixture is left to stand for hours at The solution is washed with dilute hydrochloric acid and aqueous sodium carbonate, dried over sodium sulfate and the ethyl acetate is evaporated in a vacuum at The residue is washed with ether/petroleum ether (1 :2). After drying in a high vacuum at 40 S-benzyl-B-mercaptopropionyl L-phenylalanyl- L-phenylalanine methyl ester, having a melting point of 135, [a] =-21 (dimethylformamide), is obtained.

(d) S-benzyl-p-mercaptopropionyl-L-phenylalanyl-L- phenylalanine hydrazide 52 g. of S-benzyl-B-mercaptopropionyl-L-phenylalanyl- L-phenylalanine methyl ester are dissolved in 260 cc. of anhydrous methanol, 32 cc. of anhydrous hydrazine are added and the mixture is left to stand for 15 hours at 20. The precipitate is filtered with suction and washed with methanol. After drying in a vacuum at 50, S-benzyl-B- mercaptopropionyl L phenylalanyl-L-phenylalanine hydrazide, having a melting point of 229, [a] 27 (0.3 N HCl in 95% acetic acid), is obtained.

(e) S benzyl B mercaptopropionyl-L-phenylalanyl-L- phenylalanyl L glutaminyl-L-asparaginyl-S-benzyl-L- cysteinyl-L-prolyl-G-tosyl L-arginyl-glycinamide 31 g. of S-benzyl-B-mercaptopropionyl-L-phenylalanyl- L-phenylalanine hydrazide are dissolved in a mixture of 250 ml. of dimethylformamide, 250 ml. of isopropanol and 32 cc. of 6 N hydrochloric acid, cooling is effected at 5 and 12.5 ml. of a 5 N solution of sodium nitrite in water are added while stirring. After 5 minutes the resulting solution is poured into 1.6 litres of a 0.25 N solution of sodium carbonate in water. The precipitate which forms is filtered with suction, washed with water, dried in a high vacuum at 2 and a solution of 50 g. of L-glutaminyl-L-asparaginyl S benzyl L-cysteinyl-L-prolyl-G-tosyl-L-arginylglycinamide in 300 ml. of dimethylformamide is added. The mixture is left to stand for 2 days, 1200 cc. of ethyl acetate are subsequently added and the precipitate is washed with ethyl acetate. After drying at 30 the product is washed with warm methanol. S-benzyl- S-mercaptopropionyl L phenylalanyl-L-phenylalanyl-L-glutaminyl-L- asparaginyl S benzyl L cysteinyl-L-prolyl-G-tosyl-L- arginyl-glycinamide, having a melting point of 224, [a] =37 (dimethylfor-mamide), is obtained.

6 (f) B Mercaptopropionyl L phenylalanyl L-phenylalanyl L glutaminyl L asparaginyl-L-cysteinyl-L- prolyl-L-arginyl-glycinamide The necessary amount of sodium or potassium metal is added to a solution of 5 g. of S-benzyl-B-mercaptopropionyl L phenylalanyl L phenylalanyl-L-glutaminyl- L-asparaginyl S benzyl-L-cysteinyl-L-prolyl-G-tosyl-L- arginyl-glycinamide in 1200 cc. of dry liquid ammonia to give a stable blue coloration. After the addition of 1.5 g. of ammonium chloride, the solution is evaporated to dryness. The residue contains S-mercaptopropionyl-L-phenylalanyl-L-pheuylalanylL-glutaminyl L asparaginyl L- cysteinyl-L-prolyl-L-arginyl-glycinamide and may be worked up further as such.

(g) Polypeptide compound I The residue obtained in (f) above is dissolved in 4 litres of 0.01 N acetic acid and is oxidized at a pH value of 6.59.0 by the addition of 7.5 cc. of an N solution of hydrogen peroxide in water. The pH value of the solution is adjusted to 4.5 by the addition of dilute hydrochloric acid and after the addition of 50 g. of sodium chloride or 0.34 g. of methanesulfonic acid or 0.406 g. of trifluoroacetic acid, evaporation to dryness is effected, whereby a dry powder results which keeps Well. It may be stored and when used it may be dissolved to give a clear solution. However, the solution may also be used as such, if desired after diluting with water or a salt solution.

For purposes of removing the inorganic salts, the above powder, obtained after the addition of trifluoroacetic acid, may be subjected to counter-current distribution in the system secondazy butanol/water/glacial acetic acid 120: 160: 1. After 200 transfer stages the substance is present in tubes 50 to with a maximum in tube 64 (K=0.47). After evaporation, the active polypeptide is obtained with a good yield in the form of a hygroscopic acetate, with uniform behavior in chromatography and electrophoresis. Migration in paper electrophoresis at a pH value of 5.8 and 40 v./cm.: 24 mm. in 60 minutes (the histidine used as reference migrates 55 mm.). Migration in paper electrophoresis at a pH value of 1.9 and 40 v./cm.: 38 mm. in 60 minutes (the tryptophane used as reference migrates 72 mm.). Rf in paper chromatography in the system isoamyl alcohol/pyridine/water 35:35:35:0.42. Total hydrolysis (16 hours, 6 N HCl) yields two equivalents of phenylalanine and one equivalent of each of the following amino acids: glutaminic acid, asparaginic acid, proline, arginine and glycine, and the different disulfides of cysteine and S-mercaptopropionic acid.

EXAMPLE 2 The same procedure as in Example 1 is used, except that final oxidation is effected at 035 ly the addition of 7.1 cc. of a N solution of potassium ferricyanide in water at a pH value of 5 .09.0.

EXAMPLE 3 The same procedure as in Example 1 is used, except that final oxidation is effected at 0-35" by the addition of 1.05 cc. of 1,2-diiodoethane dissolved in acetone, at a pH value of 5.5-8.5. After oxidation the excess of 1,2 diiodoethane is extracted with ethyl acetate. The ethyl acetate remaining in the aqueous solution is removed in a vacuum at 20.

7 8 What is claimed is: in which optically active amino acids have the Leon- 1. A compound selected from the group consisting of figuration; and pharmaceutically acceptable acid addition a compound of formula: salts thereof.

References Cited Hope et al., J. Biol. Chem. 237, 3146-3147 (1962).

Huguenin et al. I, Helv. Chim. Acta 45, 1629 (1962).

Huguenin et a1. II, Experientia 21, 6869 (1965).

Sturmer et 211., Experientia 21, 583-585 (1965).

LEWIS GOTTS, Primary Examiner.

M. M. KASSENOFF, Assistant Examiner.

US. Cl. X.R. 

